JP2021121221A - Polymerase variants and uses thereof - Google Patents

Abstract

To provide modified DNA polymerases (e.g., mutants) based on directed evolution experiments designed to select mutations that confer advantageous phenotypes under conditions used in industrial or research applications, e.g., catalyzing incorporation of modified polyphosphate nucleotides, e.g., tagged nucleotides, under high salt concentrations.SOLUTION: There is provided a modified DNA polymerase having DNA polymerase activity comprising an amino acid sequence having at least 95% sequence identity to an amino acid sequence of a parental polymerase represented by a specific sequence, where the amino acid sequence has substitution of T651Y substitution and P542E substitution.SELECTED DRAWING: None

Description

Related application

[001] This application is a "Polymerase Vari" filed on February 1, 2016.
US Provisional Patent Application No. 62 / 161,571, which is a partial continuation application of US Patent Application No. 15 / 012,317 entitled "ants" and is filed on May 14, 2015, entitled "Polymerase Variants", US Provisional Patent Application No. 62 / 202,895, entitled "Polymerase Variants," filed August 9, 2015, and US Patent, entitled "Polymerase Variants and Uses Thereof," filed May 10, 2016. Claim priority under Application No. 15 / 151,364.
Sequence listing
[002] This application includes a sequence listing electronically filed in ASCII format, which is incorporated herein by reference in its entirety. The above ASCII copy made on May 10, 2016 is available at 20-04338.521 WO2_SL. It is named txt and has a size of 17,989 bytes.

[003] herein, modified DNA polymerases containing mutation-based amino acid changes identified in directed evolutionary experiments designed to select more suitable enzymes, especially for application in recombinant DNA technology. offer.

[004] DNA polymerase is a family of enzymes that use single-stranded DNA as a template to synthesize complementary DNA strands. In particular, DNA polymerase can add free nucleotides to the 3'end of the newly formed strand, resulting in the extension of the new strand in the 5'to 3'direction. Most DNA polymerases are multifunctional proteins that have both polymerization and exonuclease activity. For example, many DNA polymerases have 3'→ 5'exonuclease activity. These polymerases can recognize misintegrated nucleotides, and the enzyme's 3'→ 5'exonuclease activity removes the wrong nucleotide (this activity is known as proofreading). After excision of the nucleotide, the polymerase can reinsert the correct nucleotide and replication can continue. Many DNA polymerases also have 5'→ 3'exonuclease activity.

[005] Polymerase has been found to be used in application examples of recombinant DNA, including nanopore sequencing. However, DNA strands rapidly pass through nanopores at a rate of 1 μs to 5 μs per base. This makes recording difficult, susceptible to background noise, and makes it impossible to obtain single nucleotide resolution. Therefore, in sequencing DNA strands or fragments thereof, detectable tags may be used for nucleotides. Thus, not only was the need to control the rate of sequencing DNA, but improvements (compared to wild-type enzymes) such as incorporating modified nucleotides, such as nucleotide polyphosphates with or without tags. There is a need to provide a polymerase with properties.

[006] The present invention provides mutations that provide an advantageous phenotype under conditions used in industrial or research applications, such as catalyzing the incorporation of modified nucleotide polyphosphoric acid, such as tagged nucleotides, at high salt concentrations. Provided are modified DNA polymerases (eg, mutants) based on directed evolutionary experiments designed to select.

[007] In one embodiment, H223, N of SEQ ID NO: 2 (Pol6 (with His tag)).
224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P. N545, Q546, A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648 There are variant polymerases containing at least one change at positions corresponding to I652, K655, W656, D657, V658, H660, F662, and L690.

[008] In one embodiment, a modified DNA polymerase having DNA polymerase activity, at least 70%, at least 75%, at least 80%, at least 90%, or at least the amino acid sequence set forth in SEQ ID NO: 1 or 2. Provided is a modified DNA polymerase having an amino acid sequence having 95% sequence identity.

[009] In the second embodiment, it is a modified DNA polymerase having DNA polymerase activity, H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367. , P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, I550, N552, G555, F558, A596, G603, A610, V15 , C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, and L690, and combinations thereof. Amino acids having at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2 having one or more selected amino acid substitutions. A modified DNA polymerase having a sequence is provided. In a further embodiment, the one or more amino acid substitutions are H223A, N224Y / L / Q / M / R / K, Y225L / T / I / F / A / M, H227P / E / F / Y, I295W. / F / M / E, Y342L / F, T343N / F, I357G / L / Q / H / W / M / A / E / Y / P, S360G / E / Y, L361M / W / V / F, I363V / A / R / M / W, S365Q / W / M / A / G, S366A / L, Y367L / E / M / P / N / F, P368G, D417P, E475D, Y476V, F478L, K518Q, H527W / R / L / Y / T / M, T529M / F, M531H / Y / A / K / R / W / T / L / V / G, N535L / Y / M / K / I / R / W / Q, G539Y / F, P542E / S / G, N545K / D / S / L / R / Q / W, Q546W / F, A547M / Y / W / F / V / S, L549Q / Y / H / G / R / K , I550A / W / T / G / F / S, N552L / M / S / T, G553S / T / E / Q / K / R / M, F558P / T, A596S, G603T / A / L, A610T / E , V615A / T, Y622A / M, C623G / S / Y / F, D624F / K, I628Y / V / F / L / M, Y629W / H / M / K, R632L / C, N635D, M641L / Y, A643L , I644H / M / Y, T647G / A / E / K / S / Y, I648K / R / V / N / T / L, T651Y / F / M, I652Q / G / S / N / F / T / A / L / E / K / M, K655G / F / E / N / Q / M / A, W656E, D657R / P / A / E, V658L, H660A / Y, F662I / L, L690M and combinations thereof Will be done. Modified DNA polymerases with one or more amino acid substitutions have enzyme activity, fidelity, processing power, elongation rate, sequencing accuracy, long-term continuity decoding, stability, and solubility compared to the parent polymerase. Has altered properties selected from. In one embodiment, the altered property is enzyme activity. In one embodiment, the altered property is fidelity. In one embodiment, the altered property is processing power. In one embodiment, the altered property is the elongation rate. In one embodiment, the altered property is stability. In one embodiment, the altered property is solubility. In one embodiment, the altered property is the ability to bind and / or incorporate nucleotide polyphosphate. Nucleotide polyphosphates are, for example, nucleotide tetraphosphates, nucleotide pentaphosphates, nucleotide hexaphosphates, nucleotide heptaphosphates, or nucleotide octaphosphates.

[0010] In a third embodiment, modifications having altered properties selected from enzyme activity, fidelity, throughput, elongation rate, stability, or solubility when compared to SEQ ID NO: 1 or 2. A DNA polymerase is provided. In one embodiment, the altered property is enzyme activity. In one embodiment, the altered property is fidelity. In one embodiment, the altered property is processing power. In one embodiment, the altered property is the elongation rate. In one embodiment, the altered property is stability. In one embodiment, the altered property is solubility.

[0011] In the fourth embodiment, it is a modified DNA polymerase having DNA polymerase activity, H223A, N224Y / L / Q / M / R / K, Y225L / T / I / F / A, H227P / E. / F / Y, I295W / F / M / E, Y342L / F, T343N / F, I357G / L / Q / H / W / M / A / E / Y / P, S360G / E / Y, L361M / W / V / F, I363V / A / R / M / W, S365Q / W / M / A / G, S366A / L, Y367L / E / M / P / N / F, P368G, D417P, E475D, Y476V, F478L , K518Q, H527W / R / L / Y / T / M, T529M / F, M531H / Y / A / K / R / W / T / L / V / G, N535L / Y / M / K / I / R / W / Q, G539Y / F, P542E / S / G, N545K / D / S / L / R / Q / W, Q546W / F, A547M / Y / W / F / V / S, L549Q / Y / H / G / R / K, I550A / W / T / G / F / S, N552L / M / S / T, G553S / T / E / Q / K / R / M, F558P / T, A596S, G603T / A / L, A610T / E, V615A / T, Y622A / M, C623G / S / Y / F, D624F / K, I628Y / V / F / L / M, Y629W / H / M / K, R632L / C, N635D , M641L / Y, A643L, I644H / M / Y, T647G / A / E / K / S / Y, I648K / R / V / N / T / L, T651Y / F / M, I652Q / G / S / N / F / T / A / L / E / K / M, K655G / F / E / N / Q / M / A, W656E, D657R / P / A / E, V658L, H660A / Y, F662I / L, L690M And at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% of the amino acid sequence set forth in SEQ ID NO: 1 containing one or more amino acid substitutions selected from the group consisting of and combinations thereof. Having an amino acid sequence with identity, one or more amino acid substitutions have enzyme activity, fidelity, processing power, elongation rate, sequencing accuracy, long-term continuity decoding ability, and stability compared to the parent polymerase. Provided are modified DNA polymerases that alter sex or solubility. In one embodiment, the altered property is enzyme activity. In one embodiment, the altered property is fidelity. In one embodiment, the altered property is processing power. In one embodiment, the altered property is the elongation rate. In one embodiment, the altered property is stability. In one embodiment, the altered property is solubility. In one embodiment, the altered property is the ability to bind and / or incorporate nucleotide polyphosphate. Nucleotide polyphosphates are, for example, nucleotide tetraphosphates, nucleotide pentaphosphates, nucleotide hexaphosphates, nucleotide heptaphosphates, or nucleotide octaphosphates.

[0012] In one embodiment, a variant polymerase having altered enzymatic activity as compared to SEQ ID NO: 1 or 2
a. H223A;
b. N224Y / L / Q / M / R / K;
c. Y225L / I / T / F / A / M;
d. H227P / E / F / Y;
e. I295F / E / M / W;
f. Y342L / F;
g. T343N / F;
h. I357G / L / Q / H / W / M / A / E / Y / P;
i. S360G / E / Y;
j. L361M / W / V / F;
k. I363V / A / R / M / W;
l. S365Q / W / M / A / G;
m. S366A / L;
n. Y367L / E / M / P / N / F;
o. P368G;
p. D417P;
q. E475D;
r. Y476V;
s. F478L;
t. K518Q;
u. H527W / R / L / Y / T / M;
v. T529M / F;
w. M531H / Y / A / K / R / W / T / L / V / G;
x. N535L / Y / M / K / I / R / W / Q;
y. P542E / S / G;
z. N545D / K / S / L / R / Q / W;
aa. Q546W / F;
bb. A547F / M / W / Y / V / S;
cc. L549H / Y / Q / G / R / K;
dd. I550A / W;
ee. I550T / G / F / S;
ff. N552L / M / T;
gg. G553S / T / E / Q / K / R / M;
hh. F558P / T;
ii. A596S;
JJ. G603T / A / L;
kk. A610T / E;
ll. V615A / T;
mm. Y622A / M;
nn. C623G / S / Y / A / F;
oo. D624F / K;
pp. I628Y / V / F / L / M;
qq. Y629W / H / M / K;
rr. R632L / C;
ss. N635D;
tt. M641L / Y;
uu. A643L;
vv. I644H / M / Y;
ww. T647G / A / E / K / S / Y;
xx. I648K / R / V / N / T / L;
yy. T651Y / F / M;
zz. I652Q / G / S / N / F / T / A / L / E / K / M;
aaa. K655G / F / E / N / Q / M / A;
bbb. W656E;
ccc. D657R / P / A / E;
ddd. V658L;
ee. H660A / Y;
fff. F662I / L;
ggg. L690M;
hh. S366A + N535L;
iii. T651Y + N535L;
jJJ. Y342L + E475D + F478L;
kkk. T343N + D417P + K518Q;
lll. N535L + N545K + T651Y;
mmm. I363V + E475D + Y476V;
nnn. S366L + G553S + F558P;
oo. S366A + N535L + A547M;
ppp. S366A + P542E + N545K;
qqq. S366A + P542E + I652Q;
rrr. S366A + N535L + T529M;
sss. S366A + N535L + I652Q;
ttt. S366A + N535L + N545K;
uu. T651Y + P542E + N545K;
vvv. T651Y + P542E + Q546W;
www. T651Y + P542E + S366A;
xxx. T651Y + N535L + N545K;
yy. S366A + N535I + I652Q;
zzz. T651Y + S366A + A547F;
aaaa. T647G + A547F + Y225T;
bbbb. A547F + A610T + S366A;
cccc. A547F + A610T + Y225I;
dddd. S366A + T647G + A547F;
eeee. T529M + S366A + A547F;
ffff. T647E + S366A + A547F;
gggg. T529M + T647G + A547F;
hhhh. N545K + S366A + A547F;
iii. T647G + A547F + T529M;
jjjj. T529M + A610T + A547F;
kkkkk. M641Y + T529M + A547F;
llll. T647G + C623G + A547F;
mmmm. A610T + I295W + T651Y;
nnnn. V615A + M531Y + T647G;
oooo. S366L + F478L + A596S + L690M;
pppp. H223A + G553S + A643L + F662I;
qqqq. N535L + N545K + T651Y + T529M;
rrrr. N535L + N545K + T651Y + N635D;
ssss. N535L + N545K + T651Y + I652Q;
tttt. S366A + N535L + I652Q + T529M;
uuu. S366A + S365A + P368G + G603T;
vvvv. N535L + N545K + T651Y + T647G;
www. S366A + N535L + I652Q + A547Y;
xxxx. S366A + N535L + A547M + T647G;
yyyy. T529M + S366A + A547F + N545K;
zzzz. T529M + S366A + A547F + N545R;
aaaaaa. T529M + S366A + A547F + N552L;
bbbbbb. T529M + S366A + A547F + Y629W;
cccccc. N535I + N545K + T651Y + T529M;
dddddd. N535I + N545K + T651Y + N635D;
eeee. N535I + N545K + T651Y + I652Q;
fffff. N535L + N545K + T651Y + T647G + C623G;
gggggg. N535L + N545K + T651Y + T647G + I628Y;
hhhh. S366A + N535L + A547M + T647G + S360G;
iiiiii. N535I + N545K + T651Y + I652Q + Y225I;
jjjjj. N535L + N545K + T651Y + T647G + K655G;
kkkkkk. N535L + N545K + T651Y + T647G + L549Q;
lllll. S366A + N535L + I652Q + A547Y + K655G;
mmmmm. T529M + S366A + A547F + N545L + Y629W;
nnnnnn. T529M + S366A + A547F + N545L + Y225L;
OOOO. T529M + S366A + A547F + N545L + Y225F;
pppppp. T529M + S366A + A547F + N545L + K655F;
qqqqq. T529M + S366A + A547F + N545L + N552L;
rrrrr. T529M + S366A + A547F + N545R + M531A;
ssss. T529M + S366A + A547F + N545R + G539Y;
ttttt. T529M + S366A + A547F + N545R + V658L;
uuuu. T529M + S366A + A547F + N545L + Y225L + D657R;
vvvvv. T529M + S366A + A547F + N545L + Y225L + N552L;
wwwww. T529M + S366A + A547F + N545L + Y225L + I652G;
xxxxxx. T529M + S366A + A547F + N545L + Y225L + I652Q;
yyyy. T529M + S366A + A547F + N545L + Y225L + N552M
zzzzz. T529M + S366A + A547F + N545L + Y225L + D657R + N224R;
aaaaaaa. T529M + S366A + A547F + N545L + Y225L + D657R + I628M;
bbbbbb. T529M + S366A + A547F + N545L + Y225L + D657R + K655A; and cccccc. T529M + S366A + A547F + N545L + Y225L + D657R + Y629W
Is selected from. In one embodiment, the altered property is enzyme activity. In one embodiment, the altered property is fidelity. In one embodiment, the altered property is processing power. In one embodiment, the altered property is the elongation rate. In one embodiment, the altered property is stability. In one embodiment, the altered property is solubility. In one embodiment, the altered property is the ability to bind and / or incorporate nucleotide polyphosphate. Nucleotide polyphosphates are, for example, nucleotide tetraphosphates, nucleotide pentaphosphates, nucleotide hexaphosphates, nucleotide heptaphosphates, or nucleotide octaphosphates.

[0013] In some embodiments, SEQ ID NO: 2 with the N535I + N545K + T651Y + N635D mutation, or variant polymerase with altered enzymatic activity as compared to SEQ ID NO: 1 or 2,
a. A547F + A610T + Y225I;
b. Y225T + T647G + A547F;
c. S366A + T647G + A547F;
d. S366A + A547F + A610T;
e. T529M + S366A + A547F;
f. T529M + T647G + A547F;
g. T529M + A610T + A547F;
h. N545K + S366A + A547F;
i. N545K + T647G + A547F;
j. A610T + I295W + T651Y;
k. V615A + M531Y + T647G;
l. M641Y + T529M + A547F;
m. T647E + S366A + A547F;
n. T647G + A547F + T529M;
o. T647G + C623G + A547F; and p. T651Y + S366A + A547F.
Is selected from.

[0014] In some embodiments, the variant polymerase is
a. N535L + N545K + T651Y;
b. S366A + N535L + I652Q;
c. S366A + T529M + N535L;
d. S366A + N535L + N545K;
e. S366A + N535L + A547M;
f. S366A + P542E + I652Q;
g. S366A + P542E + N545K;
h. S366A + P542E + T651Y;
i. P542E + N545K + T651Y;
j. P542E + Q546W + T651Y;
k. N535L + T651Y;
l. S366A + N535L;
m. N535L + N545K + T651Y + T529M;
n. N535L + N545K + T651Y + N635D;
o. N535L + N545K + T651Y + I652Q;
p. S366A + N535L + I652Q + T529M;
q. N535L + N545K + T651Y + T647G;
r. S366A + N535L + I652Q + A547Y;
s. S366A + N535L + A547M + T647G;
t. S366A + N535I + I652Q;
u. N535I + N545K + T651Y + T529M;
v. N535I + N545K + T651Y + N635D;
w. N535I + N545K + T651Y + I652Q;
x. N535L + N545K + T651Y + T647G + C623G;
y. N535L + N545K + T651Y + T647G + I628Y;
z. S366A + N535L + A547M + T647G + S360G;
aa. N535I + N545K + T651Y + I652Q + Y225I;
bb. N535L + N545K + T651Y + T647G + K655G;
cc. N535L + N545K + T651Y + T647G + L549Q;
dd. S366A + N535L + I652Q + A547Y + K655G;
ee. T647G + A547F + Y225T;
ff. A547F + A610T + S366A;
gg. A547F + A610T + Y225I;
hh. S366A + T647G + A547F;
ii. T651Y + S366A + A547F;
JJ. T529M + S366A + A547F;
kk. T647E + S366A + A547F;
ll. T529M + T647G + A547F;
mm. N545K + S366A + A547F;
nn. T647G + A547F + T529M;
oo. N545K + T647G + A547F;
pp. T529M + A610T + A547F;
qq. M641Y + T529M + A547F;
rr. T647G + C623G + A547F;
ss. A610T + I295W + T651Y;
tt. V615A + M531Y + T647G;
uu. T529M + S366A + A547F + N545K;
vv. T529M + S366A + A547F + N545R;
ww. T529M + S366A + A547F + N552L;
xx. T529M + S366A + A547F + Y629W;
yy. T529M + S366A + A547F + N545L + Y629W;
zz. T529M + S366A + A547F + N545L + Y225L;
aaa. T529M + S366A + A547F + N545L + Y225F;
bbb. T529M + S366A + A547F + N545L + K655F;
ccc. T529M + S366A + A547F + N545L + N552L;
ddd. T529M + S366A + A547F + N545R + M531A;
ee. T529M + S366A + A547F + N545R + G539Y;
fff. T529M + S366A + A547F + N545R + V658L;
ggg. T529M + S366A + A547F + N545L + Y225L + D657R;
hh. T529M + S366A + A547F + N545L + Y225L + N552L;
iii. T529M + S366A + A547F + N545L + Y225L + I652G;
jJJ. T529M + S366A + A547F + N545L + Y225L + I652Q;
kkk. T529M + S366A + A547F + N545L + Y225L + N552Mlll. T529M + S366A + A547F + N545L + Y225L + D657R + N224R;
mmm. T529M + S366A + A547F + N545L + Y225L + D657R + I628M;
nnn. T529M + S366A + A547F + N545L + Y225L + D657R + K655A; and oo. T529M + S366A + A547F + N545L + Y225L + D657R + Y629W
Is selected from. In some embodiments, the variant polymerase has altered enzymatic activity as compared to SEQ ID NO: 1 or 2, or the parent polymerase.

[0015] In some embodiments, SEQ ID NO: 2 with the S366A + T529M + N545L + A547F mutation, or variant polymerase with altered enzymatic activity as compared to SEQ ID NO: 1 or 2,
a. Y225L / F / A / M;
b. M531A / G;
c. G539Y;
d. N552L / T;
e. Y629W / K;
f. K655F
Is selected from. In one embodiment, the altered property is enzyme activity. In one embodiment, the altered property is fidelity. In one embodiment, the altered property is processing power. In one embodiment, the altered property is the elongation rate. In one embodiment, the altered property is stability. In one embodiment, the altered property is solubility. In one embodiment, the altered property is the ability to bind and / or incorporate nucleotide polyphosphate. Nucleotide polyphosphates are, for example, nucleotide tetraphosphates, nucleotide pentaphosphates, nucleotide hexaphosphates, nucleotide heptaphosphates, or nucleotide octaphosphates.

[0016] In some embodiments, the parent polymerase is wild-type Pol6 (SEQ ID NO: 1). In some embodiments, the parent polymerase is Pol6 (SEQ ID NO: 2) containing a His tag. In some embodiments, the parent polymerase comprises a S366A + T529M + A547F + N545L / R mutation. In some embodiments, the parent polymerase may be SEQ ID NO: 1 containing one or more mutations. For example, S366A + T529M + A547F + N545R is obtained by using S366A + T529M + A547F as a parent polymerase and then adding N545R.

[0017] In some embodiments, the modified polymerase has a larger _kchem than the parent polymerase. In some embodiments, the modified polymerase has less k _off than the parent polymerases. In some embodiments, the modified polymerase is at least 1.5 times than the parent polymerase has 2.0 times, or 2.5 times greater _k chem _{/ k off} (i.e., ratio).

[0018] Other objects, features, and advantages of the present invention will become apparent from the detailed description below. However, although detailed description and examples show preferred embodiments of the invention, this detailed description will reveal to those skilled in the art various changes and modifications within the scope and intent of the invention. It should be understood that the concrete examples are provided merely as explanations.

[0019] FIG. 6 shows an exemplary template used in a substitution assay. It relates to Example 3. [0020] FIG. 6 is a schematic diagram of the _kchem assay used herein to measure the rate of incorporation of polyphosphoric acid. It relates to Example 6. [0021] FIG. 5 is an overview of a _{fluorescence quenching based koff} assay used herein to measure the velocity properties of variant polymerases. It relates to Example 4. [0022] FIG. 5 shows an illustration of a _{fluorescently polarized koff assay and an exemplary data trace.} It relates to Example 5. [0023] It is a graph which shows the representative data of a variant polymerase from a substitution assay. It relates to Example 3. [0024] FIG. 6 is a graph of representative data of two variant polymerases from a _{fluorescently polarized koff assay.} It relates to Example 5. [0025] double layer of the upper and lower _{Hepes7.5 20mM, NaCl300mM, CaCl 2 3mM} , and in TCEP5mM, by alpha hemolysin nanopore linked to Pol6 (S366A + N535L + I652Q) -DNA complexes, static tagged thymine nucleotides at 100mV It is a figure which shows the trace of the capture. The vertical axis is the% aperture channel current (normalized), and the horizontal axis is the time in seconds. It relates to Example 8. [0026] Static capture experiments at 100 mV using a Pol6 (S366A + N535L + I652Q) -DNA complex linked to alpha hemolysin nanopores at Hepes pH 7.5 20 mM, NaCl 300 mM, CaCl 23 mM, and TCEP 5 mM above and below the bilayer. It is a graph of the plot of residence time vs. current. The average residence time for each capture of dTNP-tagged nucleotides is 1.2 seconds. It relates to Example 8. [0027] A graph of representative data of variant polymerases from a _{fluorescence quenching based kchem assay (see FIG. 2).} A dual complex of preformed polymerase and fluorescein-DNA template is rapidly mixed with saturated concentrations of dCnP-Alexa555 in the presence of ^{Mg 2+ using a Kintek stopped flow device.} Monitor the fluorescence of fluorescein over a period of time. _{The k chem} estimated from the rate-determining step is 0.2s-1. The x-axis is time (T) in seconds and the y-axis is relative fluorescence unit (RFU). [0028] is a graph of representative data of variant polymerases from a _{fluorescence quenching based koff assay (see FIG. 3).} A preformed ternary complex of polymerase, fluorescein-DNA template, and dCnP-Alexa555 was ^{preincubated in the presence of Ca 2+} and followed with excess undenatured dCTP. Fluorescein fluorescence was monitored over a period of time. _{K off} were measured from this is a 0.028s-1. The x-axis is time (T) in seconds and the y-axis is relative fluorescence unit (RFU). [0029] It is a photograph of a gel showing an augmentation by-product of the rolling circle assay. The left and right lanes are molecular ladders. The second lane from the left is at zero. All other lanes are at 40 minutes of various polymerase hits. It relates to Example 9. [0030] FIG. 6 shows a sequencing trace showing current changes that provide a record of tagged nucleotides when tagged nucleotides are incorporated into a growing DNA strand. Template DNA sequences and convened sequences of newborn strands showing> 70% accuracy are also shown (SEQ ID NOS: 6-8, respectively, in order of appearance). It relates to Example 10.

Hereinafter, the present invention will be described in detail by way of reference only, using the following definitions and examples. All patents and publications referenced herein are expressly incorporated herein by reference, including any sequences disclosed in such patents and publications.

[0032] Unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Have. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd Edition, John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER Many common dictionaries of are given to those skilled in the art. Any method and material that is similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, but preferred methods and materials are described. Regarding the definitions and terms in the art, Sambrook et al., 1989, and Ausubel FM et al., 1993 are particularly suitable for practitioners. It should be understood that the invention is not limited to the particular methods, protocols and reagents described, as the methods, protocols and reagents can vary.

[0033] The range of numbers includes numbers that define the range. The term about is used herein to mean plus or minus 10 percent (10%) of the value. For example, "about 100" refers to any number between 90 and 110.

[0034] Unless otherwise indicated, nucleic acids are written from left to right in the 5'to 3'direction, and amino acid sequences are written from left to right in the amino to carboxy direction, respectively.
[0035] The headings presented herein are not intended to limit the various aspects or embodiments of the invention, and the invention may be understood by reference in its entirety. Therefore, the terms defined directly below are more fully defined by reference in the specification as a whole. Definition:
[0036] Amino Acids: As used herein, the term "amino acid" broadly refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, the amino acid has the general structure _{H 2 N-C (H)} (R) -COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid, in some embodiments the amino acid is a D-amino acid, and in some embodiments the amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than standard amino acids, whether synthetically prepared or derived from natural sources. As used herein, "synthetic amino acids" include, but are not limited to, chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and / or substituents. Amino acids, including carboxy-terminal amino acids and / or amino-terminal amino acids of peptides, are modified by methylation, amidation, acetylation, and / or substitution with other chemicals without adversely affecting their activity. be able to. Amino acids can be involved in disulfide bonds. The term "amino acid" is used interchangeably with "amino acid residues" and can refer to free amino acids and / or amino acid residues of peptides. Whether the term refers to a free amino acid or a peptide residue is clear from the context in which the term is used. It should be noted that in the present specification, all amino acid residue sequences are represented by a formula in which the left and right directions are the conventional directions from the amino terminus to the carboxy terminus.

[0037] Base Pair (bp): As used herein, base pair is the association of adenine (A) and thymine (T), or cytosine (C) and guanine (G), in a double-stranded DNA molecule. Point to.

Complementarity: As used herein, the term "complementary" refers to a broad concept of sequence complementarity between regions of two polynucleotide chains by base pairing or between two nucleotides. Point. It is known that adenine nucleotides can form specific hydrogen bonds (“base pairing”) with nucleotides that are thymine or uracil. Similarly, it is known that cytosine nucleotides can base pair with guanine nucleotides.

[0039] DNA Binding Affinity: As used herein, the term "DNA binding affinity" typically refers to the activity of a DNA polymerase in binding to a DNA nucleic acid. In some embodiments, DNA binding activity can be measured in a two-band shift assay. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3rd Edition, Cold Spring Harbor Laboratory Press, NY), 9.63-9.75 (described for nucleic acid terminal labeling). Binding buffer to prepare a reaction mixture containing a polypeptide of at least about 0.5μg in (sodium phosphate buffer (pH 8.0) 50 mM, glycerol _{10%, KCl25mM, MgCl 2 25mM} ) about 10 [mu] l. The reaction mixture is heated to 37 ° C. for 10 minutes. The labeled double-stranded nucleic acid from about ^{1 × 10 4 ~5 × 10 4} cpm ( or about 0.5～2Ng) was added to the reaction mixture and incubated for an additional 10 minutes. The reaction mixture is placed on an unmodified polyacrylamide gel in 0.5-fold trisboric acid buffer. The reaction mixture is electrophoresed at room temperature. The gel is dried and submitted to autoradiography using standard methods. If there is a detectable decrease in the mobility of the labeled double-stranded nucleic acid, it indicates that a binding complex is formed between the polypeptide and the double-stranded nucleic acid. Such nucleic acid binding activity can be quantified by measuring the amount of radioactivity in the binding complex compared to the total amount of radioactivity in the initial reaction mixture using standard concentration measurements. .. Other methods for measuring DNA binding affinity are known in the art (see, eg, Kong et al. (1993) J. Biol. Chem. 268 (3): 1965-1975).

[0040] Elongation Rate: As used herein, the term "elongation rate" refers to the average rate at which DNA polymerase stretches a polymer strand. As used herein, high elongation rates are greater than 2 nt / s (eg, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100). , 105, 110, 115, 120, 125, 130, 135, 140 nt / s). As used in this application, the terms "extension speed", "extension speed", and "incorporation speed" are used interchangeably.

Enzymatic activity: As used herein, the term "enzymatic activity" refers to the specificity and efficiency of DNA polymerase. The enzymatic activity of DNA polymerase is also referred to as "polymerase activity" and typically refers to the activity of DNA polymerase in catalyzing template-specific synthesis of polynucleotides. The enzymatic activity of the polymerase can be measured using a variety of techniques and methods known in the art. For example, serial dilutions of the polymerase can be prepared in dilution buffers (eg, Tris.Cl, pH 8.0 20 mM, KCl 50 mM, NP40 0.5%, and Tween-20 0.5%). Removed 5μl of each _{dilution, TAPS (pH9.25) 25mM, KCl50mM} , MgCl 2 2mM, dATP0.2mM, dGTP0.2mM, dTTP0.2mM, dCTP0.1mM, activation DNA12.5μg, [α- ³² P] It can be added to 100 μM of dCTP (0.05 μCi / nmol) and 45 μl of the reaction mixture containing sterile deionized water. The reaction mixture can be incubated at 37 ° C. (or 74 ° C. for thermostable DNA polymerase) for 10 minutes, then the reaction can be quenched to 4 ° C. and stopped by adding 10 μl of ice-cooled 60 mM EDTA. 25 μl of aliquot can be removed from each reaction mixture. The non-incorporated radiolabeled dCTP can be removed from each aliquot by gel filtration (Centri-Sep, Princeton Separations, Adelfia, NJ). The column eluate can be mixed with the scintillation solution (1 ml). A scintillation counter is used to quantify the radioactivity in the column eluate to determine the amount of product synthesized by the polymerase. One unit of polymerase activity can be defined as the amount of polymerase required to synthesize 10 n mol of product in 30 minutes (Lawyer et al. (1989) J. Biol. Chem. 264: 6427-647). Other methods of measuring polymerase activity are known in the art (see, eg, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3rd Edition, Cold Spring Harbor Laboratory Press, NY)).

Purified: As used herein, "purified" is at a concentration of at least 90% by weight, or at least 95% by weight, or at least 98% by weight of the sample in which the molecule is contained. It means that it is present in the sample.

[0043] Isolated: An "isolated" molecule is usually a nucleic acid molecule that has been isolated, for example, from at least one other molecule that is associated in the natural environment of the molecule. An isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that normally expresses the nucleic acid molecule, which is located extrachromosomally or at a chromosomal position different from its natural chromosomal position. ..

% Homogeneity: As used herein, the term "% homology" is used interchangeably with the term "% homogeneity" herein and is any one of the polypeptides of the invention. Refers to the level of identity of a nucleic acid or amino acid sequence when aligned using a sequence alignment program between the nucleic acid sequences encoding the above, or the amino acid sequences of the polypeptides of the invention.

[0045] For example, as used herein, 80% homology means the same as 80% sequence identity as determined by a defined algorithm, and thus the homology of a given sequence is the place. Given sequence has more than 80% sequence identity over a length. An exemplary level of sequence identity is 80, 85, 90, 95, 98% or more of a given sequence, eg, a coding sequence of any one of the polypeptides of the invention described herein. Identity is mentioned, but not limited to.

[0046] As an exemplary computer program that can be used to determine the identity between two sequences, a series of BLAST programs published on the Internet, such as BLASTN, BLASTX, and TBLASTX, BLASTP. , And BLASTN, but are not limited to these. See also Altschul et al., 1990, and Altschul et al., 1997.

[0047] Sequence lookups are typically performed using the BLASTN program when evaluating a given nucleic acid sequence against the DNA sequence of GenBank and the nucleic acid sequences of other public databases. The BLASTX program is preferred to search for nucleic acid sequences that have been translated in all reading frames against GenBank protein sequences and amino acid sequences in other public databases. Both BLASTN and BLASTX run using the BLASTUM-62 matrix with default parameters of 11.0 open gap penalty and 1.0 extended gap penalty. (See, for example, Altschul, SF et al., Nucleic Acids Res. 25: 3389-3402, 1997.).
[0048] The preferred alignment of the selected sequences for determining "% identity" between two or more sequences is, for example, using the Clustal-W program of MacVector version 13.0.7, 10 It is run using a default parameter with an open gap penalty of .0, an extended gap penalty of 0.1, and a BLOSUM30 similarity matrix.

Modified DNA Polymerase: As used herein, the term "modified DNA polymerase" originates from another (ie, parent) DNA polymerase and is associated with one or more amino acids as compared to the parent DNA polymerase. Refers to a DNA polymerase that contains a change (eg, amino acid substitution, deletion or insertion). In some embodiments, the modified DNA polymerases of the invention originate from, or have been modified from, naturally occurring DNA polymerases, or wild-type DNA polymerases. In some embodiments, the modified DNA polymerases of the invention result from or are modified from recombinant DNA polymerases or engineered DNA polymerases. Recombinant DNA polymerases or engineered DNA polymerases include, but are not limited to, chimeric DNA polymerases, fused DNA polymerases, or other modified DNA polymerases. Typically, the modified DNA polymerase has at least one modified phenotype compared to the parent polymerase.

[0050] Mutation: As used herein, the term "mutation" refers to a change introduced into a parent sequence, including substitutions, insertions, deletions (including truncation). , Not limited to these. The consequences of the mutation include, but are not limited to, the creation of new properties, traits, functions, phenotypes, or traits not found in the protein encoded by the parent sequence.

Mutant: As used herein, the term "mutant" refers to a modified protein that exhibits altered properties when compared to the parent protein. The terms "variant" and "mutant" are used interchangeably herein.

Wild-type: As used herein, the term "wild-type" refers to a gene or gene product that, when isolated from a naturally occurring source, has the properties of that gene or gene product. ..

Fidelity: As used herein, the term "fidelity" refers to the accuracy of DNA polymerization by a template-dependent DNA polymerase, or the correct nucleotide k for the wrong nucleotide bound to the template DNA. Refers to any of the measured differences of _off. The fidelity of DNA polymerase is typically measured by error rate (the frequency of incorporation of inaccurate nucleotides, ie, nucleotides that are not incorporated in a template-dependent manner). The accuracy or fidelity of DNA polymerization is maintained by both the polymerase activity of DNA polymerase and the 3'-5'exonuclease activity. The term "high fidelity" refers to 4.45 x ^10-6 mutations / nt / less than doubling (eg 4.0 x ^10-6 , 3.5 x ^10-6 , 3.0 x ^10-6 , 2). .5 × ^10-6 , 2.0 × ^10-6 , 1.5 × ^10-6 , 1.0 × ^10-6 , 0.5 × ^10-6 mutation / nt / less than doubling) .. The fidelity or error rate of DNA polymerase can be measured using assays known in the art. For example, the error rate of DNA polymerase can be tested as described herein, or Johnson et al., Biochim Biophys Acta. May 2010; 1804 (5): 1041-1048 can be tested as described.

Nanopores: As used herein, the term "nanopores" generally refers to pores, channels, or passages formed in or otherwise provided to the membrane. The membrane may be an organic membrane such as a lipid bilayer or a synthetic membrane such as a membrane formed of a polymer material. The membrane may be a polymer material. The nanopores may be placed adjacent to or near a detection circuit such as, for example, a complementary metal oxide semiconductor (CMOS) circuit or a field effect transistor (FET) circuit, or an electrode connected to the detection circuit. In some examples, nanopores have characteristic widths or diameters in the order of 0.1 nanometers (nm) to about 1000 nm. Some nanopores are proteins. Alpha hemolysin, MspA, is an example of protein nanopores.

[0055] Nucleotides: As used herein, a monomeric unit of DNA or RNA consists of a sugar moiety (pentose), a phosphate, and a nitrogen-containing heterocyclic base. The base is linked to the sugar moiety via the carbon of the glycoside (1'carbon of pentose), and the combination of the base and the sugar is a nucleoside. If the nucleoside contains a phosphate group attached to the 3'or 5'position of the pentose, it is called a nucleotide. Sequences of operatively linked nucleotides are typically referred to herein as "base sequences" or "nucleotide sequences", where the left-to-right direction is 5'end to 3 'Represented by an equation that is in the traditional direction to the end. As used herein, "modified nucleotide" refers to nucleotide polyphosphoric acid, such as nucleotides 3, 4, 5, 6, 7, or octaphosphoric acid.

Oligonucleotides or polynucleotides: As used herein, the term "oligonucleotide" is defined as a molecule that comprises two or more, preferably more than three deoxyribonucleotides and / or ribonucleotides. Its exact size will depend on many factors, which in turn will depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be obtained synthetically or by cloning. As used herein, the term "polynucleotide" refers to a polymer molecule composed of nucleotide monomers covalently bonded within a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides.

Polymerase: As used herein, "polymerase" refers to an enzyme that catalyzes the polymerization of nucleotides (ie, polymerase activity). Normally, the enzyme initiates synthesis at the 3'end of the primer annealed to the template sequence of the polynucleotide and proceeds to the 5'end of the template strand. "DNA polymerase" catalyzes the polymerization of deoxynucleotides.

Primer: As used herein, the term "primer" is suitable when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain, eg, at a suitable temperature. In the presence of four different nucleotide primers and thermostable enzymes in a flexible buffer (“buffer” includes pH, ionic strength, cofactors, etc.), it can serve as a starting point for nucleic acid synthesis. Refers to an oligonucleotide that can be naturally occurring or synthetically produced. The primer is preferably single-stranded in order to maximize amplification efficiency, but may be double-stranded instead. In the case of double strands, the primer is first treated to separate the strands and then used to prepare the extension product. The primer is preferably an oligodeoxyribonucleotide. The primer must be long enough to be the starting material for the synthesis of the extension product in the presence of the thermostable enzyme. The exact length of the primer will depend on many factors, including temperature, primer source, and application of the method. For example, depending on the complexity of the target sequence, oligonucleotide primers typically contain 15-25 nucleotides, but can contain more or less nucleotides. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.

[0059] Processing Capacity: As used herein, "processing capacity" refers to the ability of a polymerase to remain attached to a template and perform multiple modification reactions. "Modification reactions" include, but are not limited to, polymerization and exonuclease cleavage. In some embodiments, "processing capacity" refers to the ability of a DNA polymerase to carry out a series of polymerization steps without the enzyme dissociating from the growing DNA strand in the middle. Typically, the "processing capacity" of a DNA polymerase is the length of the nucleotide in which the DNA polymerase is polymerized or modified from the growing DNA strand without premature dissociation (eg, 20 nt, 300 nt, 0.5 to 0.5 nt). 1 kb or more). "Processing capacity" can depend on the nature of the polymerase, the sequence of the DNA template, and the reaction conditions, such as salt concentration, temperature, or the presence of a particular protein. As used herein, the term "high throughput" refers to more than 20 nt per association / dissociation with a template (eg, more than 40 nt, more than 60 nt, more than 80 nt, more than 100 nt, more than 120 nt, more than 140 nt, more than 160 nt). , Over 180 nt, over 200 nt, over 220 nt, over 240 nt, over 260 nt, over 280 nt, over 300 nt, over 320 nt, over 340 nt, over 360 nt, over 380 nt, over 400 nt, or more). Processing power can be measured according to the methods defined herein and WO01 / 92501A1 (MJ Bioworks, Inc., Applied Nucleic Acid Modifying Enzymes, published December 06, 2001).

Synthesis: As used herein, the term "synthesis" is used in a template-dependent manner to create new polynucleotide strands or extend existing polynucleotides (ie, DNA or RNA). Refers to any in vitro method to be performed. Synthesis according to the invention involves amplification using a polymerase to increase the number of replicates of the polynucleotide template sequence. As a result of the synthesis (eg, amplification) of the polynucleotide, the nucleotide is incorporated into the polynucleotide (ie, primer), thereby forming a new polynucleotide molecule complementary to the polynucleotide template. The formed polynucleotide molecule and its template can be used as a template for synthesizing further polynucleotide molecules. As used herein, "DNA synthesis" includes, but is not limited to, PCR, polynucleotide labeling (ie, for probes and oligonucleotide primers), and polynucleotide sequencing.

[0061] Template DNA molecule: As used herein, the term "template DNA molecule" refers to a nucleic acid strand in which a complementary nucleic acid strand is synthesized by DNA polymerase, for example, in a primer extension reaction.

[0062] Template-Dependent Method: As used herein, the term "template-dependent method" refers to a process involving the extension of a template-dependent primer molecule (eg, DNA synthesis by DNA polymerase). .. The term "template-dependent method" is typically used for RNA or DNA polynucleotides in which the sequence of the newly synthesized polynucleotide chain is defined by well-known rules for complementary base pairing. Refers to synthesis (see, eg, Watson, JD et al., Molecular Biologic of the Gene, 4th Edition, WA Benjamin, Inc., Menlo Park, Calif. (1987)).

[0063] Tags: As used herein, the term "tag" refers to a detectable portion that may be an atom or molecule, or a group of atoms or molecules. Tags can exhibit optical, electrochemical, magnetic, or electrostatic (eg, inductive, capacitive) properties, the properties of which can be detected utilizing nanopores.

[0064] Tagged Nucleotides: As used herein, the term "tagged nucleotides" refers to tagged or modified nucleotides. The tag may be covalently attached to a sugar, phosphoric acid (or polyphosphoric acid), or base. The tag may be on the terminal phosphate.

Vector: As used herein, the term "vector" refers to a nucleic acid structure designed for transfer between different host cells. "Expression vector" refers to a vector capable of incorporating and expressing a heterologous DNA fragment in an exogenous cell. Many prokaryotic expression vectors and eukaryotic expression vectors are commercially available. Selection of an appropriate expression vector is known to those of skill in the art.

[0066] The polymerase variants shown herein are WO2013 / 188841 (Genia Technologies, Inc., Chip Set-Up and).
It is useful for chip-based polynucleotide sequencing as described in High-Accuracy Nucleic Acid Sequence, published December 19, 2013).

[0067] Desirable properties of the polymerase used for DNA sequencing are:
a. Slow _koff (for modified nucleotides)
b. Fast _{k on} (against modified nucleotides)
c. High fidelity d. Low exonuclease activity e. DNA strand substitution f. Faster _kchem (for modified nucleotide substrates)
g. Improved stability h. Processing capacity i. Salt tolerance j. Compatibility of mounting on nanopores k. Ability to incorporate nucleotide polyphosphates with 4, 5, 6, 7, or 8 phosphates, such as nucleotide tetraphosphate, nucleotide pentaphosphate, nucleotide hexaphosphate, nucleotide heptaphosphate, or nucleotide octaphosphate. .. Sequencing accuracy m. Long lead length, ie long continuous decoding.
Nomenclature
[0068] As used herein and in the claims, the conventional one-letter and three-letter notation of amino acid residues is used.

[0069] For ease of reference, the polymerase variants of this application are described using the following nomenclature.
[0070] Original amino acid (s): Position (s): Substituted amino acid (s). According to this nomenclature, for example, the replacement of serin with alanine at position 242 is
Ser242Ala or S242A
Shown as.

[0071] Multiple mutations are separated by a plus sign. That is,
Ala30Asp + Glu34Ser or A30N + E34S
Represents mutations at positions 30 and 34 that replace alanine and glutamic acid with asparagine and serine, respectively.

[0072] Where one or more alternative amino acid residues can be inserted at a given position, they are indicated as A30N / E, or A30N or A30E.
[0073] Unless otherwise stated, the residue numbers correspond to the numbering of residues in SEQ ID NO: 2.
Polymerase site-specific mutagenesis
[0074] Crostridium phage phiCPV4 wild-type sequences are shown herein (SEQ ID NO: 3, nucleic acid coding region and His tag; SEQ ID NO: 1, protein coding region) and are available elsewhere (National Center). for Bioinformatics or GenBank receipt number AFH27113).

[0075] Point mutations can be introduced using the QuickChange Lighting2 kit (Stategene / Agilent) according to the manufacturer's instructions.

[0076] Primers can be ordered from commercial companies such as IDT DNA.
Nanopore construction and insertion
[0077] The method described herein can use nanopores with a polymerase attached to the nanopores. In some cases, it is desirable to have only one polymerase per nanopore (eg, so that only one nucleic acid molecule is sequenced in each nanopore). However, many nanopores, including, for example, alpha hemolysin (aHL), can be multimeric proteins with multiple subunits (eg, 7 subunits in aHL). The subunits can be the same replica of the same polypeptide. As used herein, a multimeric protein (eg, nanopore) having a defined ratio of a modified subunit (eg, a-HL variant) to an unmodified subunit (eg, a-HL) is provided. Also provided herein are methods of making multimeric proteins (eg, nanopores) having a defined ratio of modified subunits to unmodified subunits.

[0078] With reference to FIG. 27 of WO2014 / 074727 (Genia Technologies, Inc.), the method of constructing a protein having a plurality of subunits is to prepare a plurality of first subunits and to prepare a plurality of first subunits. The second subunit has been modified as compared to the first subunit, including the provision of two subunits 2710. In some cases, the first subunit is wild-type (eg, purified from natural sources or recombinantly made). The second subunit can be modified in any suitable way. In some cases, the second subunit has a attached protein (eg, a polymerase) (eg, as a fusion protein).

[0079] The modified subunit can include a chemically reactive moiety (eg, an azide group or alkyne group suitable for forming a link). In some cases, the method further comprises performing a reaction (eg, Click chemical addition cyclization) to attach a real entity (eg, polymerase) to the chemically reactive moiety.

[0080] The method involves contacting the first subunit with the second subunit 2715 in a first ratio to form a plurality of proteins 2720 with the first subunit and the second subunit. Can be further included. For example, one portion of the modified aHL subunit, which has a reactive group suitable for attaching a polymerase, can be mixed with six parts of the wild-type aHL subunit (ie, the first ratio is 1: 6). Multiple proteins can have multiple ratios of the first subunit to the second subunit. For example, the mixed subunits can form several nanopores with a stoichiometric ratio of modified subunits: unmodified subunits (eg, 1: 6, 2: 5, 3: 4). ..

[0081] In some cases, proteins are formed by simply mixing subunits. For example, in the case of aHL nanopores, a surfactant (eg, deoxycholic acid) can cause the aHL monomer to adopt a pore structure. Nanopores are lipids (eg, 1,2-difitanoyl-sn-glycero-3-phosphocholine (DPhPC) or 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DoPhPC)), and medium temperature (eg, DoPhPC). , Less than about 100 ° C.). In some cases, mixing DPhPC with a buffer solution produces a large multimembrane vesicle (LMV), to which the aHL subunit is added and the mixture is incubated at 40 ° C. for 30 minutes to form pores. ..

[0082] When two different subunits (eg, a natural wild-type protein and a second aHL monomer that can contain a single point mutation) are used, the resulting protein will be (eg, wild-type). Can have mixed stoichiometric ratios (of mutant proteins). The stoichiometric ratio of these proteins can follow an equation that depends on the concentration ratio of the two proteins used in the pore formation reaction. This formula is as follows.
100P _m = 100 [n! / M! (Nm)! ] ・ F _mut ^m・ f _wt ^{n to} m, probability of pores having P _m = m mutant subunits in the formula n = total number of subunits (for example, 7 in aHL)
m = Number of “mutant” subunits f _mut = Percentage or ratio of mutant subunits _{mixed together f wt} = Percentage or ratio of wild-type subunits mixed together
[0083] The method can further include fractionating multiple proteins to increase the proportion of protein having a second ratio of the first subunit to the second subunit 2725. For example, a nanopore protein with only one modified subunit can be isolated (eg, the second ratio is 1: 6). However, either second ratio is suitable. The distribution of the second ratio can also be fractionated, for example increasing the proportion of proteins with either one or two modified subunits. As shown in FIG. 27 of WO2014 / 074727, the total number of subunits forming a protein is not always 7 (eg, different nanopores can be used, or 6 subunits can be used. Alpha hemolysin nanopores with can form). In some cases, the proportion of proteins with only one modified subunit is increased. In such cases, the second ratio is one second subunit per first subunit (n-1), where n is the number of subunits that make up the protein.

[0084] The first ratio can be the same as the second ratio, but it is not required. In some cases, proteins with mutant monomers can form with lower efficiency than proteins without mutant subunits. In this case, the first ratio can be greater than the second ratio (eg, preferred if a second ratio of mutant subunit 1: non-mutant subunit 6 is desired in the nanopores. It may be necessary to mix subunits in a ratio greater than 1: 6 to form a large number of 1: 6 proteins).

[0085] Proteins with different second subunit ratios can behave differently (eg, have different retention times) in separation. In some cases, proteins are fractionated using chromatography such as ion exchange chromatography or affinity chromatography. Since the first and second subunits can be identical except for modification, the number of modifications in the protein can serve as a basis for separation. In some cases, either the first or second subunit has a purification tag (eg, in addition to modification) to allow fractionation or improve fractionation efficiency. .. In some cases, polyhistidine tags (His tags), streptavidin tags (Strep tags), or other peptide tags are used. In some examples, the first and second subunits each contain a different tag, and the fractionation step is fractionated based on each tag. In the case of His tags, the tags are charged at low pH (histidine residues are positively charged below the pKa of the side chain). Due to the significant difference in charge in one of the aHL molecules compared to the other, using ion exchange chromatography, 0, 1, 2, 3, 4, 5, 6, or 7 "charges" Oligomers with the "tagged" aHL subunit can be separated. In principle, this charge tag can be a string of arbitrary amino acids carrying a constant charge. 28 and 29 show examples of nanopore fractions based on His tags. FIG. 28 shows a plot of UV absorbance at 280 nanometers, UV absorbance at 260 nanometers, and conductivity. Peaks correspond to nanopores with varying ratios of modified and unmodified subunits. FIG. 29 of WO2014 / 074727 shows a fraction of aHL nanopores and variants thereof using both His and Strep tags.

[0086] In some cases, after fractionation, a real entity (eg, polymerase) is attached to the protein. The protein can be a nanopore and the real thing can be a polymerase. In some examples, the method further comprises inserting a protein with a subunit of the second ratio into the double layer.

[0087] In some situations, the nanopore can contain multiple subunits. The polymerase can be attached to one of the subunits, with at least one and less than all of the subunits containing a first purification tag. In some examples, nanopores are alpha hemolysin or variants thereof. In some examples, all of the subunits include a first purification tag or a second purification tag. The first purification tag can be a polyhistidine tag (eg, in the subunit with a polymerase).
Polymerase attached to the nanopore
[0088] In some cases, the polymerase (eg, DNA polymerase) is attached to and / or placed in the vicinity of the nanopore. The polymerase can be attached to the nanopores before or after the nanopores are incorporated into the membrane. In some examples, nanopores and polymerases are fusion proteins (ie, single polypeptide chains).

[0089] The polymerase can be attached to the nanopores in any suitable manner. In some cases, the polymerase is attached to a nanopore (eg, hemolysin) protein monomer, followed by a polymerase (eg, for 6 nanopore (eg, hemolysin) monomers without a polymerase). Construct the entire nanopore heptamer (with the ratio of one monomer with). The nanopore heptamer can then be inserted into the membrane.

Another method of attaching the polymerase to the nanopore is to attach a linker molecule to the hemolysin monomer, or to mutate the hemolysin monomer to have an attachment site, and then (eg, a linker). It involves constructing an entire nanopore heptamer (in the ratio of one monomer with a linker and / or attachment site to six hemolysin monomers without and / or attachment sites). The polymerase can then be applied to the attachment site or attachment linker (eg, in large quantities prior to insertion into the membrane). Polymerases can also be attached to attachment sites or attachment linkers after forming nanopores (eg, heptamers) on the membrane. In some cases, multiple nanopore-polymerase pairs are inserted into multiple membranes of the biochip (eg, placed in wells and / or electrodes). In some examples, attachment of the polymerase to the nanopore complex is performed on the biochip on each electrode.

[0091] The polymerase can be attached to the nanopores using any suitable chemistry (eg, covalent and / or linker). In some cases, the polymerase is attached to the nanopores with molecular staples. In some examples, molecular staples contain three amino acid sequences (indicated by linkers A, B, and C). The linker A can extend from the hemolysin monomer and the linker B can extend from the polymerase, where the linker C wraps the linker A and the linker B (eg, both the linker A and the linker B). By doing so, the polymerase can be connected to the nanopore. Linker C can also be constructed to be part of linker A or linker B, thus reducing the number of linker molecules.

[0092] In some examples, the polymerase is linked to nanopores using Solulink ™ chemistry. Solulink ™ can be a reaction between HyNic (6-hydrazino-nicotinic acid, aromatic hydrazine) and 4FB (4-formylbenzoic acid ester, aromatic aldehyde). In some examples, the polymerase uses Click chemistry (eg, available from Life Technologies) to ligate the nanopores. In some cases, a zinc finger mutation is introduced into the hemolysin molecule and then a molecule (eg, a DNA intermediate molecule) is used to ligate the polymerase to the zinc finger site of the hemolysin.

Other linkers that can be used to attach the polymerase to the nanopore are direct gene linkages (eg, (GGGGS) _1-3 (SEQ ID NO: 4) amino acid linkers), transglutaminase-mediated linkages (eg, RSKLG). (SEQ ID NO: 5)), sortase-mediated linkage, and cysteine-modified chemical linkage. Specific linkers considered useful herein are (GGGGS) _1-3 (SEQ ID NO: 4), N-terminal K-tag (RSKLG (SEQ ID NO: 5)), ΔTEV site (12-25), ΔTEV site + N-terminus of SpyCatcher (12-49).
Device setup
[0094] Nanopores may be formed on a film arranged adjacent to a detection electrode in a detection circuit such as an integrated circuit, or may be embedded in other ways. The integrated circuit may be an application specific integrated circuit (ASIC). In some examples, the integrated circuit is a field effect transistor or complementary metal oxide semiconductor (CMOS). The detection circuit can be located on a chip or other device with nanopores, or can be located outside the chip or device, for example in an off-chip arrangement. The semiconductor can be any semiconductor, including, but not limited to, group IV semiconductors (eg, silicon) and group III-V semiconductors (eg, gallium arsenide). See, for example, WO 2013/123450 for device and device setup for detecting nucleotides or tags.

[0095] A sensor using pores (eg, a biochip) can be used for single molecule electron matching. A sensor using pores can include nanopores of the present disclosure formed on a membrane located adjacent to or near the detection electrode. The sensor can include a counter electrode. The membrane includes a transformer side (ie, the side facing the detection electrode) and a cis side (ie, the side facing the counter electrode).

[0096] In subsequent disclosures relating to experiments, the following abbreviations apply: eq (equivalent); M (molar concentration); μM (micromolar concentration); N (specified); mol (molar); mmol ( Mmol); μmol (micromol); nmol (nanomolar); g (gram); mg (milligram); kg (kilogram); μg (microgram); L (liter); ml (milliliter); μl (microliter) Cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C. (degrees of degrees); h (hours); min (minutes); sec (seconds); msec (milliseconds) ..

[0097] The present invention will be further described in the following examples. The examples are not intended to limit the scope of the claims in any way. The accompanying figures are intended to be viewed as part of the specification and description of the present invention. All references are expressly incorporated herein by reference in their entirety. The following examples are provided for illustration purposes only and are not intended to limit the claimed invention.

Directional mutagenesis
[0098] This example illustrates the introduction of a mutation into a pol6 polymerase at a desired location.

[0099] DNA encoding the His-tagged wild-type pol6 was purchased from a commercial source (DNA 2.0, Menlo Park, California). The sequence was confirmed by sequencing.

[00100] Polymerase was expressed as is for mutant screening (N-terminal Hi).
s-Pol6). The SpyCatcher domain was manipulated and placed at the N-terminus or C-terminus of pol6 to test that pol attaches to the chip.

[00101] Reasonable positions affecting Pol6-nucleotide binding were identified based on known crystal structure homology modeling.
[00102] For initial screening, each of the rational positions was placed in Gly, Ala, Leu, Glu, Gln, Lys, His, Tyr, Pro, Trp, Thr, or Met using the Q5 mutagenesis protocol. Mutated.

[00103] Primers for each mutagenesis reaction were designed using the NEB base changer protocol and ordered from IDT in 96-well plate format.
[00104] Forward and reverse primers were 5'phosphorylated in high throughput (HTP) format using T4 polynucleotide kinase (PNK) purchased from NEB. A typical 25 μl reactant contained 15 μl of 10 μM primer, 5 μl of 5-fold reaction buffer (from NEB), 1.25 μl of PNK enzyme, and 3.75 μl of water. The reaction was carried out at 37 ° C. for 30 minutes and the enzyme was heat inactivated at 65 ° C. for 20 minutes.

[00105] PCR mutagenesis was performed using Q5 DNA polymerase from NEB. Typical 25 μl reactants are Q5 buffer 5 μl, GC enhancer 5 μl, 10 mM dNTP 0.5 μl, 10 μM phosphorylation mutagenesis forward and reverse primer 1.25 μl, Q5 polymerase 0.25 μl, and 5 ng / ml wild type. Pol6 mold, i.e. containing His-Pol6 1μl, and _H 2 _O10.75μl.

[00106] When PCR was complete, 0.5 μl of Dpn1 was added to 25 μl of the PCR mix and incubated at 37 ° C. for 1 hour.
[00107] Add 2.5 μl of Dpn1 treated PCR product to 2.5 μl of Blunt / TA ligase master mix. Incubated for 1 hour at room temperature.

[00108] 1 μl of ligation mix, 96-well BL21DE3 cells (EMD)
Add 20 μl of Millipore) and incubate on ice for 5 minutes.
[00109] Using a PCR device, heat shock at 42 ° C. for exactly 30 seconds and place on ice for 2 minutes.

[00110] Add 80 μl of SOC and incubate in an incubator at 37 ° C. for 1 hour without shaking.
[00111] Add 100 μl of SOC or ultrapure water, and add 50 to 100 μg / ml of kanamycin.
Sow on a 48-well LB agar plate containing.

Expression and purification
[00112] The following examples detail how the pol6 variant was expressed and purified using the high-throughput method.

[00113] DNA encoding the variant in the pD441 vector (expression plasmid) was transformed into E. coli competent cells to prepare a glycerol stock. Starting with traces taken from the glycerol stock, 1 ml of starter culture was grown in LB containing 0.2% glucose and 100 μg / ml kanamycin for about 8 hours. Add 25 μl of logarithmic starter culture to 1 ml of 96 deepwell plate expression medium (terrific bouillon (TB) self-inducing medium supplemented with 0.2% glucose, 50 mM potassium phosphate, 5 mM MgCl, and 100 μg / ml kanamycin). Transfer. The plates were incubated at 28 ° C. for 36-40 hours with shaking at 250-300 rpm.

[00114] The cells were then collected by centrifugation at 3200 xg for 30 minutes at 4 ° C. 200 μl of lysis buffer (potassium phosphate 20 mM pH 7.5, NaCl 100 mM, Tween 20 0.5%, TCEP 5 mM, imidazole 10 mM, PMSF 1 mM, 1 × Bug Buster, lysozyme 100 μg / ml, decanted to remove medium and pre-cooled cell pellet. And protease inhibitors) and incubated for 20 minutes at room temperature with gentle stirring. 20 μl from the 10-fold stock is then added to a final concentration of 100 μg / ml DNase, 5 mM MgCl2, 100 μg / ml RNase I and incubated on ice for 5-10 minutes to generate a lysate. Add 200 μl of 1 M potassium phosphate and pH 7.5 (final concentration is about 0.5 M potassium phosphate in 400 μl of solution) to the solution, and centrifuge at 4 ° C. for 10 minutes at about 1500 rpm. This allows filtration with a Pall filter plate (part number 5053, 3 micron filter). The clear lysate was then placed in an equilibrated 96-well His-Pur cobalt plate (Pierce Part No. 90995) and allowed to bond for 15-30 minutes.

[00115] The effluent (FT) was collected by centrifugation at 500 x G for 3 minutes. The FT was then washed 3 times with 400 μl wash buffer 1 (potassium phosphate 0.5M pH 7.5, NaCl 1M, TCEP 5 mM, imidazole 20 mM, and Tween 20 0.5%). The FT was then washed twice with 400 μl wash buffer 2 (Tris 50 mM pH 7.4, KCl 200 mM, TCEP 5 mM, Tween 20 0.5%, imidazole 20 mM).

[00116] Pol was added to 200 μl of elution buffer (Tris 50 mM pH 7.4, KCl).
Elution was carried out using 200 mM, TCEP 5 mM, Tween 20 0.5%, imidazole 300 mM, glycerol 25%) and collected after 1-2 minutes of incubation. The eluate is re-inserted into the same His-Pur plate 2-3 times to obtain Pol6 concentrated in the eluent. The purified polymerase is more than 95% pure as assessed by SDS-PAGE. The protein concentration is about 3 uM (0.35 mg / ml) as assessed by Nanodrop, with a 260/280 ratio of 0.6.

[00117] Polymerase activity is confirmed by fluorescence substitution assay (see Example 3).

Measurement of activity
[00118] This example shows a method of measuring the activity of a variant polymerase.
Substitution assay protocol
[00119] This assay characterizes the ability of mutant polymerases to integrate nucleotide polyphosphates into DNA strands and the ability of mutant polymerases to untie and replace double-stranded DNA.

[00120] Stock reagents are:
Low salt

High salt

[00121] For single and double mutant screening:
[00122] Reagent A is used as a diluent to make four different nucleotide conditions at 1.42 times.

[00123] For screening for triple mutants:
[00124] Reagent A is used as a diluent to make four different nucleotide conditions at 1.42 times.

[00125] Nucleotide condition 1 tests for activity at high concentrations of hexaphosphate.
[00126] Nucleotide condition 2 tests for activity at low concentrations of hexaphosphate.
[00127] Nucleotide condition 3 tests for misintegration rate (ie, fidelity). If the mutant polymerase exhibits significant activity in only 3 of the 4 required nucleotides, it is concluded that the mutant polymerase does not identify the correct or incorrect nucleotide during the extension of the DNA strand. do.

[00128] Nucleotide condition 4 tests for exonuclease activity. If the polymerase shows significant activity in the absence of nucleotides, we conclude that the polymerase shows exonuclease activity.

[00129] In each reaction well of the 96-well half-area transparent plate,
Reagent A / Nucleotide Mix 23 μl
Polymerase (1-10 μM) 2 μl
Add.

[00130] Shake with a plate shaker at 800 RPM for 10 minutes.
[00131] Add 5 μl of 1.4 M NaCl to each well to raise the NaCl concentration to 300 mM, or add 5 μl of 525 mM NaCl to each well to raise the NaCl concentration to 150 mM.

[00132] Incubate for 30 minutes.
[00133] In a BMG LABTECH plate reader, inject 10 μl of reagent B and read the fluorescent signal for 2-10 minutes.

Representative data from the variant polymerase substitution assay is shown in FIG.
The activity of the polymerase is A. dTnP20μM + dA, C, G3P15μM (red square; ■), B.I. dTnP5μM + dA, C, G3P15μM (blue diamond shape; ◆), C.I. In the presence of dA, C, G3P 15 μM (green triangle; ▲), or D.I. Measurements were made using a substitution assay in the absence of nucleotides (purple x). A and B indicate that the mutant variant incorporates nucleotide polyphosphate and can be extended along the DNA template with nucleotide polyphosphate. C indicates that the variant has not lost fidelity and has not incorrectly incorporated chaotic nucleotides in the absence of T nucleotides. D. Indicates that the signal generated is not the result of polymerase exonuclease activity in the absence of all nucleotides. All four curves are representative of a single variant tested on four different assay plates as part of the polymerase screening.

measurement of k _off
[00135] using the stopped-flow assay described below, of the variant polymerase k _{o
The ff} speed was measured.

[00136] In Reagent A, the polymerase is attached to a fluorescein-labeled DNA template-primer with Cy3 (or Alexa555) -linked nucleotide polyphosphoric acid in the presence of a non-catalytic divalent metal such as Ca2 +. It forms a FRET pair, fluorescein is the donor fluorophore and Cy3 is the acceptor fluorophore. Reagent B contains tracking nucleotides. In this assay, the first nucleotide incorporated into the template / primer is cytosine.

[00137] Reagent A (NaCl75 mM, HEPES25 mM (pH 7.5), CaCl2
2 mM, fluorescein-template / primer 250 nM, dCnP-Cy3 20 uM, and polymerase> 250 nM) were freshly prepared by mixing the ingredients with the final addition of the polymerase. The polymerase was incubated in Reagent A for 10 minutes.

[00138] Reagent B (NaCl75 mM, HEPES25 mM (pH 7.5), CaCl2
2 mM, and dCTP200uM) were prepared.
[00139] When reagents A and B are mixed, dCTP competes with dCnP-Cy3 for association with dC.
If the TP concentration is excessive, an increase in fluorescence is observed. The assay can be performed using either a stop-flow device (Kintek Corp) or a fluorescent plate reader. The increase in fluorescence over time by fitting to the primary or secondary exponential, obtain rate constants k _off of the particular polymerase.

[00140] Table 1 shows the purification yield and k _off for the selected variant.

[00141] See FIG. 3 for a schematic diagram of the assay and a graph of exemplary reactions. See FIG. 10 for representative data.

measurement of k _off
[00142] In this example, use of fluorescence polarization, showing an alternative method of measuring k _off.
[00143] Triton 25 mM pH 7.0, KCl 75 mM, Triton-X100 0
.. Assay master mix containing hairpin fluorescein-labeled DNA template and dC6P-C6-Cy3 tagged nucleotide 250 nM using assay buffer containing 01%, 1 x BSA (100 ug / ml), EDTA 0.5 mM, CaCl2 2 mM, DTT 2 mM. Was prepared. 55 μl of master mix was added to each well of a black 96-well Costar plate and 20 μl of polymerase variant purified from 1 ml of culture was added in high throughput (HTP) format. The plates were shaken in a plate shaker for 1 minute to form a uniform ternary complex of polymerase-DNA template-nucleotides. Plate BMG Polarstar Plate Reader (BMG LABTECH)
Inc. , North Carolina), and the target millipolarized light was adjusted to 200 mP and 10% to have a gain around 2000. The excitation filter was set to 485 nM and the emission filter was set to 590 to 20 nM. The syringe was filled with 1 ml of 1 mM dCTP tracking nucleotide solution. Data were collected with a minimum of 30 flushes per well per interval with a total start read time of 60 seconds. For hit mutants that showed slow dissociation, the flush was increased to 50 or greater and longer read times were taken. Data collection was initiated with an infusion of 25 μl of 1 mM dCTP.

[00144] See FIG. 4 for a schematic diagram of the assay and a graph of exemplary reactions.
[00145] from k _off based assay fluorescence polarization, two variants polymerase (
See FIG. 6 for representative data of S366A + N535L + I652Q (B6) and S366A + P542E + I652Q (C6)). mP is metrically polarized. The preformed ternary complex of polymerase-DNA template-dCnP-Alexa555 was followed with undenatured dCTP and polarized dCnP-Alexa555 was monitored over time.

measurement of k _chem
[00146] In this example, FR for measuring the _{kchem of the variant polymerase.}
The assay based on ET is shown.

[00147] In Reagent A, the polymerase is bound to a fluorescein-labeled DNA template-primer. Reagent B contains Cy3 (or Alexa555) -linked nucleotide polyphosphoric acid in the presence of a catalytic divalent metal such as ^{Mg 2+.} In this protocol, the first nucleotide incorporated into the template / primer is cytosine.

[00148] Reagent A (NaCl 75 mM, HEPES 25 mM (pH 7.5), fluorescein-template / primer 250 nM, polymerase> 250 nM) was prepared. The polymerase was incubated in Reagent A for 10 minutes.

[00149] Reagent B (NaCl 75 mM, HEPES 25 mM (pH 7.5), MgCl ₂
10 mM, and dCnP-Cy3 20 uM) were prepared.
[00150] When reagents A and B are mixed, the polymerase-fluorescein-template-primer complex binds to dCnP-Cy3 and the fluorescence is quenched. Mg ²⁺ allows the polymerase to incorporate nucleotides, which release the cleavage product, pyrophosphate with Cy3, nP-Cy3. Fluorescence increases as the quencher is released. The assay can be performed using either a stop-flow device (Kintek Corp) or a fluorescent plate reader.

[00151] See FIG. 2 for a schematic diagram of the assay and a graph of exemplary reactions. See FIG. 9 for representative data.

Installation on nanopores
[00152] In this example, a method of attaching a variant polymerase to a nanopore, eg, α-hemolysin, is shown.

[00153] The polymerase may be linked to the nanopores by any suitable means. For example, PCT / US2013 / 068967 (published as WO2014 / 074727; Genia Technologies, Inc.), PCT / US2005 / 909702 (published as WO2006 / 028508; Present and Fellows of Harvard College), Published as 083249; Columbia University).

[00154] A polymerase, eg, a variant pol6 DNA polymerase, was ligated to a protein nanopore (eg, alpha hemolysin) via a linker molecule. Specifically, the SpyTag and SpyCatcher systems that spontaneously form covalent isopeptide linkages under physiological conditions were used. For example, Li et al., J Mol Biol. See January 23, 2014; 426 (2): 309-17.

[00155] poly6 variant SpyCatcherHis tags were expressed according to Example 2 and purified using a cobalt affinity column. SpyCatcher polymerase and SpyTag oligomerized nanopore proteins were incubated overnight at 4 ° C. in _{3 mM SrCl 2.} The 1: 6-polymerase-template complex was then purified using molecular sieving chromatography.

[00156] A linker was attached to either the N-terminus or the C-terminus of the pol6 variant.
Variants attached at the N-terminus were found to be more robust, eg, more stable. Therefore, a linker attached to the N-terminus was used.

Activity in biochip
[00157] This example demonstrates the ability of a variant polymerase to bind a nanopore-bound variant polymerase to a tagged nucleotide, thereby allowing the detection of closed channel currents in the polymerase-attached nanopore.

[00158] Polymerase was attached to the nanopore and embedded in a lipid bilayer on the wells of a semiconductor sensor chip, also known as a biochip. The lipid bilayer was formed as described in PCT / US2014 / 061853 (filed October 22, 2014 under the title "Methods for Forming Lipid Bilayers on Biochips") and attached with a polymerase. A nanopore was inserted.

[00159] Variant polymerase was complexed with template DNA under low salt conditions.
[00160] The ability of variant polymerases bound to nanopores to bind to tagged nucleotides was measured in static capture experiments. In static capture experiments, the closed channel current is measured when the tagged nucleotide binds to the polymerase and the tagged nucleotide is presented to the nanopore. Static capture experiments are performed in the presence of Ca2 +, which interferes with DNA catalysis and elongation and makes it possible to detect repeated captures of the same type of tagged nucleotides. In this experiment, the tagged nucleotide used was a dTnP-tag.

An exemplary polymerase variant Pol6 (S366A + N535L + I652Q) linked to the alpha hemolysin nanopore of the biochip was complexed with the template DNA.
[00162] Pol6 (S366A + N535L + I652Q) ( tag is T30 (SEQ ID NO: 9)) tagged thymidine nucleotides by -DNA complexes static capture, bilayer Hepes7.5 20 mM in the vertical, NaCl300mM, CaCl ₂ 3mM , And recorded at 100 mV in the presence of TCEP 5 mM.

[00163] The results are shown in FIGS. 7 and 8. The trace in FIG. 7 shows the electrolytic current measured at 100 mV across the pores as a function of time. The opening pore current at this voltage was about 1 nA (top trace) and the closed pore current at the same voltage was about 0.33 nA (center trace). The aperture channel current was normalized to 1 according to the system software. The closed channel current was reduced by dTnP-T30 (SEQ ID NO: 9) to 33% of the open channel current. The current interruption shown in this trace is associated with the binding of thymidine polyphosphate by the variant Pol6-DNA complex that occurs near the nanopores. The corresponding top histogram in FIG. 7 (right) shows the frequency of current interruptions observed at 100 mV with a normalized current change relative to the open pore current in the same pore. The histogram (right) corresponding to the central trace shows the frequency of current interruptions observed at 100 mV with a normalized current change relative to the closed pore current in the same pore.

[00164] FIG. 8 shows the residence time of static capture of the tagged thymidine shown in FIG. FIG. 8 (left) shows a histogram of the number of occurrences of tagged dTNP binding to variant Pol6 as a function of current normalized to aperture channel current. The average residence time for each capture of dTNP-tagged nucleotides was measured to be 1.2 seconds. Background capture of tags in the pores (ie, those not mediated by polymerase) has a residence time in the range of a few milliseconds (data not shown). Since the goal of enzyme evolution was to improve the dissociation rate of tagged nucleotide polyphosphates, it can be understood that the residence time is long enough to record polymerase-mediated capture that is clearly distinguishable from the background. As shown in FIG. 8, the average dwell time of 1.2 seconds is well above the background. The cell index is a color-based scheme of about 8000 cells present on the chip used in this experiment.

[00165] Based on the data, exemplary variant polymerase Pol6 (S366A + N5)
35L + I652Q) has been shown to be able to bind to tagged nucleotides and allow detection of current changes by polymerase-tagged nanopores.

[00166] The results show that the variant polymerase attached to the nanopore of the biochip binds to the tagged nucleotide with high fidelity and allows the tagged nucleotide to be detected for sufficient time, and in some cases, eg, nucleotide integration. It provides evidence that it can be presented to nanopores with a residence time that gives sufficient time to reduce the probability of sequencing errors such as insertions and deletions during nanopore sequencing.

Rolling circle amplification assay
[00167] This example describes amplification of a polynucleotide template in a rolling circle assay.

[00168] The mold used was an in-house manufactured mold HFcirc10. The mold is a simple cyclic mold with a length of about 150 bp.
[00169] Total reaction volume 40 μl (Reagent A 28 μl + 2 μM Polymerase 2 μl + Reagent B
The assay was performed with 10 μl).

[00170] Reagent A:

[00171] Reagent B:

[00172] 2 μl of 2 μM polymerase was added to 28 μl of reagent A to make the molar ratio of DNA: polymerase (100 nM each) in 40 μl of the final assay mix 1: 1. The Reagent A / Polymerase mix was incubated for 10 minutes under this 75 mM salt condition to attach the polymerase to the DNA.

[00173] Next, 10 μl of Reagent B was added to the Reagent A / Polymerase mixture to initiate the reaction.
[00174] At predetermined time points, 10 μl of sample was removed from the reactants and added to 10 μl of formamide containing 50 mM EDTA to inactivate the reaction. Samples were taken at 0, 10, 30 and 40 minutes.

[00175] The formamide sample was heated to 94 ° C. for about 3 minutes to denature the protein and the secondary structure of the DNA. The sample was not allowed to cool to 4 ° C. 100 × SYBR GREEN or GOLD dye 2 μl was added.

[00176] 15 μl of each sample in a 1.2% agarose gel at 100 V for 1 hour 1
It was run for 5 minutes.
An image of the gel was acquired using the blue tray of the Bio-Rad GEL DOC EZ imager.

[00178] FIG. 11 shows the results of the assay at 40 minutes. Molecular ladders are shown in lanes 1 and 19 counting from left to right. Lane 2 has a sample with t = 0 and no product is visible. Each of lanes 3-18 is a different variant pol6 polymerase.

All the variants shown are capable of strand substitution and long kilobase DNA products can all be produced with nucleotide hexaphosphate.

Sequencing template DNA using tagged nucleotides
[00180] In this example, the variant polymerase is shown to be functional in synthetic sequencing on a biochip.

[00181] A of heteropolymer template using Pol6-26i-D44A polymerase
C sequencing was performed at Hepes 20 mM, pH 8, potassium glutamate 500 mM, and MgCl 23 mM at room temperature. Nanopores with polymerases were implanted in the lipid bilayer as described herein. DNA with the primer was added and complexed with the polymerase. Four different tagged nucleotides were added at a concentration of 25 μM. Sequencing by synthesis may proceed as described in WO2014 / 074727 entitled "Nucleic Acid Sequence Using Tags". The trace in FIG. 12 shows 76% sequencing accuracy for the heteropolymer template and a read length of 96 bp.

[00182] The examples and embodiments described herein are for illustrative purposes only, and various modifications or modifications that take into account the examples and embodiments will be proposed to those skilled in the art. , It will be understood that the purpose and scope of this application and the scope of the attached claims will be included. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for any purpose.

SEQ ID NO: 1-Wild-type Pol6 (DNA polymerase [Clostridium phage phiCPV4]; GenBank: AFH27113.1)
1 mdkhtqyvke hsfnydeykk anfdkiecli fdtesctnye ndntgarvyg wglgvtrnhn
061 miygqnlnqf wevcqnifnd wyhdnkhtik itktkkgfpk rkyikfpiav hnlgwdvefl
121 kyslvengfn ydkgllktvf skgapyqtvt dveepktfhi vqnnnivygc nvymdkffev
181 enkdgsttei glcldffdsy kiitcaesqf hnyvhdvdpm fykmgeeydy dtwrspthkq 241 ttlelryqyn diymlrevie qfyidglcgg elpltgmrta ssiafnvlkk mtfgeektee 301 gyinyfeldk ktkfeflrkr iemesytggy thanhkavgk tinkigcsld inssypsqma 361 ykvfpygkpv rktwgrkpkt eknevyliev gfdfvepkhe eyaldifkig avnskalspi 421 tgavsgqeyf ctnikdgkai pvykelkdtk lttnynvvlt sveyefwikh fnfgvfkkde 481 ydcfevdnle ftglkigsil yykaekgkfk pyvdhftkmk venkklgnkp ltnqakliln 541 gaygkfgtkq nkeekdlimd knglltftgs vteyegkefy rpyasfvtay grlqlwnaii 601 yavgvenfly cdtdsiycnr
evnsliedmn aigetidkti lgkwdvehvf dkfkvlgqkk 661 ymyhdckedk tdlkccglps darkiiigqg fdefylgknv egkkqrkkvi ggcllldtlf 721 tikkimf

SEQ ID NO: 2-Pol6 (with His tag)
MHHHHHHHHS GGSDKHTQYV KEHSFNYDEY KKANFDKIEC LIFDTESCTN 50
YENDNTGARV YGWGLGVTRN HNMIYGQNLN QFWEVCQNIF NDWYHDNKHT 100
IKITKTKKGF PKRKYIKFPI AVHNLGWDVE FLKYSLVENG FNYDKGLLKT 150
VFSKGAPYQT VTDVEEPKTF HIVQNNNIVY GCNVYMDKFF EVENKDGSTT 200
EIGLCLDFFD SYKIITCAES QFHNYVHDVD PMFYKMGEEY DYDTWRSPTH 250
KQTTLELRYQ YNDIYMLREV IEQFYIDGLC GGELPLTGMR TASSIAFNVL 300
KKMTFGEEKT EEGYINYFEL DKKTKFEFLR KRIEMESYTG GYTHANHKAV 350
GKTINKIGCS LDINSSYPSQ MAYKVFPYGK PVRKTWGRKP KTEKNEVYLI 400
EVGFDFVEPK HEEYALDIFK IGAVNSKALS PITGAVSGQE YFCTNIKDGK 450
AIPVYKELKD TKLTTNYNVV LTSVEYEFWI KHFNFGVFKK DEYDCFEVDN 500
LEFTGLKIGS ILYYKAEKGK FKPYVDHFTK MKVENKKLGN KPLTNQAKLI 550
LNGAYGKFGT KQNKEEKDLI MDKNGLLTFT GSVTEYEGKE FYRPYASFVT 600
AYGRLQLWNA IIYAVGVENF LYCDTDSIYC NREVNSLIED MNAIGETIDK 650
TILGKWDVEH VFDKFKVLGQ KKYMYHDCKE DKTDLKCCGL PSDARKIIIG 700
QGFDEFYLGK NVEGKKQRKK VIGGCLLLDT LFTIKKIMF * 739

SEQ ID NO: 3-His-tagged Pol6 (DNA sequence)
ATG CATCACC ATCATCATCA CCACCAC AGC GGCGGTTCCG ACAAACACAC 50
GCAGTACGTC AAAGAGCATA GCTTCAATTA TGACGAGTAT AAGAAAGCGA 100
ATTTCGACAA GATCGAGTGC CTGATCTTTG ACACCGAGAG CTGCACGAAT 150
TATGAGAACG ATAATACCGG TGCACGTGTT TACGGTTGGG GTCTTGGCGT 200
CACCCGCAAC CACAATATGA TCTACGGCCA AAATCTGAAT CAGTTTTGGG 250
AAGTATGCCA GAACATTTTC AATGATTGGT ATCACGACAA CAAACATACC 300
ATTAAGATTA CCAAGACCAA GAAAGGCTTC CCGAAACGTA AGTACATTAA 350
GTTTCCGATT GCAGTTCACA ATTTGGGCTG GGATGTTGAA TTCCTGAAGT 400
ATAGCCTGGT GGAGAATGGT TTCAATTACG ACAAGGGTCT GCTGAAAACT 450
GTTTTTAGCA AGGGTGCGCC GTACCAAACC GTGACCGATG TTGAGGAACC 500
GAAAACGTTC CATATCGTCC AGAATAACAA CATCGTTTAT GGTTGTAACG 550
TGTATATGGA CAAATTCTTT GAGGTCGAGA ACAAAGACGG CTCTACCACC 600
GAGATTGGCC TGTGCTTGGA TTTCTTCGAT AGCTATAAGA TCATCACGTG 650
TGCTGAGAGC CAGTTCCACA ATTACGTTCA TGATGTGGAT CCAATGTTCT 700
ACAAAATGGG TGAAGAGTAT GATTACGATA CTTGGCGTAG CCCGACGCAC 750
AAGCAGACCA CCCTGGAGCT GCGCTACCAA TACAATGATA TCTATATGCT 800
GCGTGAAGTC ATCGAACAGT TTTACATTGA CGGTTTATGT GGCGGCGAGC 850
TGCCGCTGAC CGGCATGCGC ACCGCTTCCA GCATTGCGTT CAACGTGCTG 900
AAAAAGATGA CCTTTGGTGA GGAAAAGACG GAAGAGGGCT ACATCAACTA 950
TTTTGAATTG GACAAGAAAA CCAAATTCGA GTTTCTGCGT AAGCGCATTG 1000
AAATGGAATC GTACACCGGT GGCTATACGC ACGCAAATCA CAAAGCCGTT 1050
GGTAAGACTA TTAACAAGAT CGGTTGCTCT TTGGACATTA ACAGCTCATA 1100
CCCTTCGCAG ATGGCGTACA AGGTCTTTCC GTATGGCAAA CCGGTTCGTA 1150
AGACCTGGGG TCGTAAACCA AAGACCGAGA AGAACGAAGT TTATCTGATT 1200
GAAGTTGGCT TTGACTTCGT GGAGCCGAAA CACGAAGAAT ACGCGCTGGA 1250
TATCTTTAAG ATTGGTGCGG TGAACTCTAA AGCGCTGAGC CCGATCACCG 1300
GCGCTGTCAG CGGTCAAGAG TATTTCTGTA CGAACATTAA AGACGGCAAA 1350
GCAATCCCGG TTTACAAAGA ACTGAAGGAC ACCAAATTGA CCACTAACTA 1400
CAATGTCGTG CTGACCAGCG TGGAGTACGA GTTCTGGATC AAACACTTCA 1450
ATTTTGGTGT GTTTAAGAAA GACGAGTACG ACTGTTTCGA AGTTGACAAT 1500
CTGGAGTTTA CGGGTCTGAA GATTGGTTCC ATTCTGTACT ACAAGGCAGA 1550
GAAAGGCAAG TTTAAACCTT ACGTGGATCA CTTCACGAAA ATGAAAGTGG 1600
AGAACAAGAA ACTGGGTAAT AAGCCGCTGA CGAATCAGGC AAAGCTGATT 1650
CTGAACGGTG CGTACGGCAA ATTCGGCACC AAACAAAACA AAGAAGAGAA 1700
AGATTTGATC ATGGATAAGA ACGGTTTGCT GACCTTCACG GGTAGCGTCA 1750
CGGAATACGA GGGTAAAGAA TTCTATCGTC CGTATGCGAG CTTCGTTACT 1800
GCCTATGGTC GCCTGCAACT GTGGAACGCG ATTATCTACG CGGTTGGTGT 1850
GGAGAATTTT CTGTACTGCG ACACCGACAG CATCTATTGT AACCGTGAAG 1900
TTAACAGCCT CATTGAGGAT ATGAACGCCA TTGGTGAAAC CATCGATAAA 1950
ACGATTCTGG GTAAATGGGA CGTGGAGCAT GTCTTTGATA AGTTTAAGGT 2000
CCTGGGCCAG AAGAAGTACA TGTATCATGA TTGCAAAGAA GATAAAACGG 2050
ACCTGAAGTG TTGCGGTCTG CCGAGCGATG CCCGTAAGAT TATCATTGGT 2100
CAAGGTTTCG ACGAGTTTTA TCTGGGCAAA AATGTCGAAG GTAAGAAGCA 2150
ACGCAAAAAA GTGATCGGCG GTTGCCTGCT GCTGGACACC CTGTTTACGA 2200
TCAAGAAAAT CATGTTCTAA 2220
Prior art documents Patent documents
[1] PCT / US2005 / 009702 (published as WO2006 / 028508 on 16 March 2006; President and Fellows of Harvard College; entitled METHODS AND APPARATUS FOR CHARACTERIZING
POLYNUCLEOTIDES).
[2] PCT / US2011 / 065640 (published as WO2012 / 083249 on 21 June 2012; Columbia University; entitled DNA SEQUENCING BY SYNTHESIS USING MODIFIED NUCLEOTIDES AND NANOPORE DETECTION).
[3] PCT / US2013 / 068967 (published as WO2014 / 074727 on 15 May 2014; Genia Technologies; entitled NUCLEIC ACID SEQUENCING USING TAGS).
[4] PCT / US2013 / 046012 (Genia Technologies, Inc., entitled CHIP SET-UP AND HIGH-ACCURACY NUCLEIC ACID SEQUENCING, published 19 Dec 2013 as WO2013 / 188841).
[5] US 2013/0053544 (Isis Innovation Limited) entitled Peptide Tag Systems That
Spontaneously Form an Irreversible Link to Protein Partners via Isopeptide Bonds, published 28 February 2013.
Non-patent literature
[1] Altschul, SF, et al., J. Mol. Biol. (1990) 215: 403-410.
[2] Altschul, SF, et al., Nucleic Acids Res. 25: 3389-3402, 1997.
[3] Ausubel, Frederick et al., (1992) Short Protocols in Molecular Biology, Current Protocols in Molecular Biology, 2nd ed., Greene Publishing Associates & John Wiley & Sons. New York, NY
[4] Gardner et al., Nucleic Acids Res. (2012) pages 1-12 (doi: 10.1093 / nar / gks330; First published online: May 8, 2012).
[5] Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991).
[6] Johnson, et al., Biochim Biophys Acta. 2010 May; 1804 (5): 1041-1048.
[7] Kong et al. (1993) J. Biol. Chem. 268 (3): 1965-1975).
[8] Lawyer et al. (1989) J. Biol. Chem. 264: 6427-647.
[9] Li et al, Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag; J Mol Biol. (2014 Jan 23) 426 (2): 309-17.
[10] Sambrook et al, ( 1989) Molecular Cloning:. A Laboratory Manual (. 2 nd ed, Cold Spring Harbor Laboratory Press, NY).
. [11] Sambrook et al ( 2001) Molecular Cloning: A Laboratory Manual (. 3 rd ed, Cold Spring Harbor Laboratory Press, NY) at 9.63-9.75 (describing end-labeling of nucleic acids).
[12] Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994).
[13] Watson, JD et al., In: Molecular Biology of the Gene, 4th Ed., WA Benjamin, Inc., Menlo Park, Calif. (1987)).
[14] Zakari and Howarth, (2010) Spontaneous Intermolecular Amide Bond Formation
between Side Chains for Irreversible Peptide Targeting, J. Am. Chem. Soc., 132 (13): 4526-4527.
[15] Zakari, B. et al., (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion, PNAS 109 (12): E690-E697.

Claims (23)

A modified DNA polymerase having DNA polymerase activity, which comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2, wherein the amino acid sequence is H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546 L549, I550, N552, G555, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655 The modified DNA polymerase comprising one or more amino acid substitutions selected from the group consisting of D657, V658, H660, F662, L690, and combinations thereof. The substitutions are H223A, N224Y / L / Q / M / R / K, Y225L / T / I / F / A / M, H227P / E / F / Y, I295W / F / M / E, Y342L / F, T343N / F, I357G / L / Q / H / W / M / A / E / Y / P, S360G / E / Y, L361M / W / V / F, I363V / A / R / M / W, S365Q / W / M / A / G, S366A / L, Y367L / E / M / P / N / F, P368G, D417P, E475D, Y476V, F478L, K518Q, H527W / R / L / Y / T / M, T529M / F, M531H / Y / A / K / R / W / T / L / V / G, N535L / Y / M / K / I / R / W / Q, G539Y / F, P542E / S / G, N545K / D / S / L / R / Q / W, Q546W / F, A547M / Y / W / F / V / S, L549Q / Y / H / G / R / K, I550A / W / T / G / F / S, N552L / M / S / T, G553S / T / E / Q / K / R / M, F558P / T, A596S, G603T / A / L, A610T / E, V615A / T, Y622A / M, C623G / S / Y / F, D624F / K, I628Y / V / F / L / M, Y629W / H / M / K, R632L / C, N635D, M641L / Y, A643L, I644H / M / Y, T647G / A / E / K / S / Y, I648K / R / V / N / T / L, T651Y / F / M, I652Q / G / S / N / F / T / A / L / E / K / M, K655G / The first aspect of claim 1, wherein the group is selected from the group consisting of F / E / N / Q / M / A, W656E, D657R / P / A / E, V658L, H660A / Y, F662I / L, L690M and combinations thereof. Modified DNA polymerase. The modified DNA polymerase according to claim 1, wherein the modified polymerase has properties that are different from those of the parent polymerase. The altered properties include fidelity, processing power, elongation rate, stability, solubility, as well as nucleotide tetraphosphate, nucleotide pentaphosphate, nucleotide hexaphosphate, nucleotide heptaphosphate, or nucleotide octaphosphate. The modified DNA polymerase according to claim 3, which is selected from the ability to bind nucleotide polyphosphate and / or to incorporate nucleotide polyphosphate. The modified DNA polymerase according to claim 3, wherein the altered property is the ability to integrate a nucleotide polyphosphoric acid having 4, 5, 6, 7, or 8 phosphates into a growing DNA strand. The modified DNA polymerase according to claim 5, wherein the nucleotide polyphosphoric acid is tagged. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with S366 A / L. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with E475D. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with F478L. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with K518Q. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with T529M. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with N535L. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with N545K / L. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with A547F. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with G553S. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with T647G / Y. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with T651Y. The modified DNA polymerase according to claim 1, wherein the modified polymerase has a substitution consistent with I652Q / A / L / E / K / M. H223A;
N224Y / L / Q / M / R / K;
Y225L / I / T / F / A / M;
H227P / E / F / Y;
I295F / E / M / W;
Y342L / F;
T343N / F;
I357G / L / Q / H / W / M / A / E / Y / P;
S360G / E / Y;
L361M / W / V / F;
I363V / A / R / M / W;
S365Q / W / M / A / G;
S366A / L;
Y367L / E / M / P / N / F;
P368G;
D417P;
E475D;
Y476V;
F478L;
K518Q;
H527W / R / L / Y / T / M;
T529M / F;
M531H / Y / A / K / R / W / T / L / V / G;
N535L / Y / M / K / I / R / W / Q;
P542E / S / G;
N545D / K / S / L / R / Q / W;
Q546W / F;
A547F / M / W / Y / V / S;
L549H / Y / Q / G / R / K;
I550A / W;
I550T / G / F / S;
N552L / M / T;
G553S / T / E / Q / K / R / M;
F558P / T;
A596S;
G603T / A / L;
A610T / E;
V615A / T;
Y622A / M;
C623G / S / Y / A / F;
D624F / K;
I628Y / V / F / L / M;
Y629W / H / M / K;
R632L / C;
N635D;
M641L / Y;
A643L;
I644H / M / Y;
T647G / A / E / K / S / Y;
I648K / R / V / N / T / L;
T651Y / F / M;
I652Q / G / S / N / F / T / A / L / E / K / M;
K655G / F / E / N / Q / M / A;
W656E;
D657R / P / A / E;
V658L;
H660A / Y;
F662I / L;
L690M;
S366A + N535L;
T651Y + N535L;
Y342L + E475D + F478L;
T343N + D417P + K518Q;
N535L + N545K + T651Y;
I363V + E475D + Y476V;
S366L + G553S + F558P;
S366A + N535L + A547M;
S366A + P542E + N545K;
S366A + P542E + I652Q;
S366A + N535L + T529M;
S366A + N535L + I652Q;
S366A + N535L + N545K;
T651Y + P542E + N545K;
T651Y + P542E + Q546W;
T651Y + P542E + S366A;
T651Y + N535L + N545K;
S366A + N535I + I652Q;
T651Y + S366A + A547F;
T647G + A547F + Y225T;
A547F + A610T + S366A;
A547F + A610T + Y225I;
S366A + T647G + A547F;
T529M + S366A + A547F;
T647E + S366A + A547F;
T529M + T647G + A547F;
N545K + S366A + A547F;
T647G + A547F + T529M;
T529M + A610T + A547F;
M641Y + T529M + A547F;
T647G + C623G + A547F;
A610T + I295W + T651Y;
V615A + M531Y + T647G;
S366L + F478L + A596S + L690M;
H223A + G553S + A643L + F662I;
N535L + N545K + T651Y + T529M;
N535L + N545K + T651Y + N635D;
N535L + N545K + T651Y + I652Q;
S366A + N535L + I652Q + T529M;
S366A + S365A + P368G + G603T;
N535L + N545K + T651Y + T647G;
S366A + N535L + I652Q + A547Y;
S366A + N535L + A547M + T647G;
T529M + S366A + A547F + N545K;
T529M + S366A + A547F + N545R;
T529M + S366A + A547F + N552L;
T529M + S366A + A547F + Y629W;
N535I + N545K + T651Y + T529M;
N535I + N545K + T651Y + N635D;
N535I + N545K + T651Y + I652Q;
N535L + N545K + T651Y + T647G + C623G;
N535L + N545K + T651Y + T647G + I628Y;
S366A + N535L + A547M + T647G + S360G;
N535I + N545K + T651Y + I652Q + Y225I;
N535L + N545K + T651Y + T647G + K655G;
N535L + N545K + T651Y + T647G + L549Q;
S366A + N535L + I652Q + A547Y + K655G;
T529M + S366A + A547F + N545L + Y629W;
T529M + S366A + A547F + N545L + Y225L;
T529M + S366A + A547F + N545L + Y225F;
T529M + S366A + A547F + N545L + K655F;
T529M + S366A + A547F + N545L + N552L;
T529M + S366A + A547F + N545R + M531A;
T529M + S366A + A547F + N545R + G539Y;
T529M + S366A + A547F + N545R + V658L;
T529M + S366A + A547F + N545L + Y225L + D657R;
T529M + S366A + A547F + N545L + Y225L + N552L;
T529M + S366A + A547F + N545L + Y225L + I652G;
T529M + S366A + A547F + N545L + Y225L + I652Q;
T529M + S366A + A547F + N545L + Y225L + N552M
T529M + S366A + A547F + N545L + Y225L + D657R + N224R;
T529M + S366A + A547F + N545L + Y225L + D657R + I628M;
T529M + S366A + A547F + N545L + Y225L + D657R + K655A; and T529M + S366A + A547F + N545L + Y225L + D657R + Y629W
The modified DNA polymerase according to claim 1, which is selected from. The modified DNA polymerase according to claim 1, which comprises S366A + T529M + N545L + A547F. a. Y225L / F / A / M;
b. M531A / G;
c. G539Y;
d. N552L / T;
e. Y629W / K;
f. K655F / Q / M / A; and g. D657R / E / P / A
The modified DNA polymerase according to claim 20, further comprising at least one mutation selected from. The modified DNA polymerase according to claim 1, which comprises S365A + S366A + P368G + G603T. The modified DNA polymerase according to claim 1, wherein the polymerase contains less than 10 mutations.

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